Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The present invention relates generally to the field of plaque amyloid deposits that are the hallmarks of Alzheimer""s disease. In particular, the invention relates to an isolated, functionally-active protein that has gamma-secretase activity. Gamma-secretase activity is necessary for amyloid production. The present invention also relates to methods for isolating integral-membrane proteins and protein complexes, including the gamma-secretase protein of the invention, and assays for detecting gamma-secretase activity.
Alzheimer""s disease is characterized by neuropathological lesions in the brain, marked by extracellular amyloid plaques in the cerebral and limbic cortices and intraneuronal paired helical filaments and neurofibrillary tangles. Commonly, Alzheimer""s disease is a disease of the elderly with incidence increasing sharply after 60 years of age. However, early onset of Alzheimer""s disease may strike patients only 40-50 years old, and is often associated with Familial Alzheimer""s disease (FAD).
The course of both types of Alzheimer""s disease appears to be the same. The major proteinaceous component of vascular and plaque amyloid deposits is the Axcex2-42 peptide which is generated by proteolytic cleavage of xcex2APP. There is extensive evidence that supports the hypothesis that the Axcex2-42 peptide plays an essential role in the pathogenesis of Alzheimer""s disease. The generation of Axcex2 peptides from the xcex2 amyloid precursor protein (xcex2APP) involves three different protease activities designated alpha-, beta-, and gamma-secretases, and is altered by mutations in xcex2APP, and two different presenilins designated PS1 and PS2. To date, nucleotide sequences have been determined for xcex2APP (Kang, J. et al., 1987 Nature 325:733-736), PS1 (Sherrington, R., et al., 1995 Nature 375:754-760), and PS2 (Levy-Lahad, E., et al., 1995 Science 269:973-977). A candidate nucleotide sequence that may encode the protein having beta-secretase activity (Vassar, R., et al., 1999 Science 286:735-741; U.S. Pat. Nos. 5,744,346 and 5,942,400), and a candidate alpha secretase molecule (Lammich, S., et al., 1999 Proc. Natl. Acad. Sci. USA 98:3922-3927) have been identified. The isolated sequence for gamma-secretase remains elusive.
The mature xcex2APP protein is an integral-membrane protein found in the plasma membrane, Golgi apparatus, and endoplasmic reticulum. The xcex2APP protein resembles a cell-surface receptor having a large extracellular N-terminal domain, a single transmembrane domain, and a small cytoplasmic C-terminal tail (Kang, J., et al., 1987 supra). Splice variants of the xcex2APP mRNA encode APP polypeptides of 770, 750, and 695 amino acids. All these forms of xcex2APP include the cleavage region and can give rise to amyloidogenic Axcex2 peptides. In normal cells, xcex2APP undergoes one of two different sequential cleavage pathways that involve alpha-, beta-, and gamma-secretases (Dovey, H. F., et al., 1993 Neuroreport 4:1039-1042; Selkoe, D. J., et al., 1994 Ann. Rev. of Cell Biol. 10:373-403; Asami-Odaka, A., et al., 1995 Biochemistry 34:10272-10278).
In one cleavage pathway, alpha-secretase cleaves xcex2APP in the extracellular, membrane/proximal domain (e.g., C-terminus to amino acid residue 687 of the 770 amino acid form of xcex2APP) to generate a soluble N-terminal fragment (e.g., the alpha-sAPP fragment) and a membrane-bound C-terminal fragment (e.g., the 9 kDa CTF or C83 CTF). Then, gamma-secretase cleaves the membrane-bound CTF, within the membrane-bound domain, to generate the p3 fragment (e.g., the 3 kDa fragment) and a 6 kDa C-terminal fragment.
In another cleavage pathway, beta-secretase cleaves xcex2APP in the extracellular, membrane-proximal domain (e.g., C-terminal to amino acid residue 671 of the 770 amino acid form of xcex2APP) to generate a soluble N-terminal fragment (e.g., the 100 kDa NTF or beta-sAPP fragment) and a membrane-bound C-terminal fragment (e.g., the 11 kDa CTF or C 100 CTF). Then, gamma-secretase cleaves the membrane-bound CTF, within the membrane-bound domain, to generate the p6 fragment (e.g., the 6 kDa fragment) and Axcex2 peptide (e.g., the 4 kDa fragment).
The amino acid sequence of the gamma-secretase cleavage region is known (Duffy, C. L., et al., 1988 Brain Res. 474:100-111; Castano, E. M. and Frangione, B. 1988 Lab. Invest. 58:122-132). Gamma-secretase cleaves at variable sites within the cleavage region (Haass, C. and Selkoe, D. J. 1993 Cell 75:1039-1042) to generate a population of Axcex2 peptides having heterogeneous C-terminal ends. In normal patients, the Axcex2 peptide is found in two predominant forms, the majority Axcex2-40 form and the minority Axcex2-42 form each having a distinct COOH-terminus. Patients with the most common form of FAD show an increase in the amount of the 42 form. The Axcex2-40 form is not associated with early deposits of amyloid plaques. In contrast, the Axcex2-42 form accumulates early and predominantly in the parenchymal plaques and there is strong evidence that Axcex2-42 plays a major role in amyloid plaque deposits in FAD patients (Roher, A. E., et al., 1993 Proc. Natl. Acad. Sci. USA 90:10836; Iwatasubo, T., et al., 1994 Neuron 13:45; Yamaguchi, H., et al., 1995 Amyloid Int. J. Clin. Invest. 2:7-16; Mann, D. M., et al., 1996 Am. J. Pathol. 148:1257).
It has been generally thought that the same gamma-secretase enzyme generates the xe2x88x9240 and xe2x88x9242 forms. To date, this question remains unsettled because researchers in the field have reported conflicting results. For example, two research groups have independently reported in vitro results which suggest certain protease inhibitors selectively decrease the levels of Axcex2-42 and concluded that Axcex2-40 and 42 are generated by two different gamma-secretases (Citron, M., et al., 1996 Proc. Nat. Acad. Sci. USA 93:13170-13175; Klafki, H. -W., et al., 1996 J. Biol. Chem. 271:28655-28659). A third research group has compared the relative ability of a series of protease inhibitors to inhibit secretion of Axcex2-40 and 42 peptides and reached the opposing conclusion that the Axcex2-40 and -42 peptides are generated by a single protease (Durkin, J. T. et al., 1999 Journal of Biological Chemistry 274:20499-20504).
The Axcex2-40 and -42 forms are secreted constitutively in a wide variety of cells/tissues, and are found as soluble forms in biological fluids (Seubert, P., et al., 1992 Nature 359:325 375; Shoji, M., et al., 1992 Science 258:126-129) thus allowing extensive analysis of both forms of the Axcex2 peptide in FAD patients. Some FAD patients have elevated levels of the Axcex2-42 peptide in their serum (Scheuner, D., et al., 1996 Nat. Med. 2:864-870). It is known that mutations in the xcex2APP, PS1 or PS2 gene, found in FAD patients, alter cleavage of the xcex2APP protein to increase the relative amount of the AD-42 peptide (Tomita, T. et al., 1997 Proc. Natl. Acad. Sci. USA 94:2025-2030; Duff, K., et al., 1996 Nature 383:710-713; Borchelt, D., et al., 1996 Neuron 17:1005-1013; Citron, M., et al., 1997 Nat. Med. 3:67-72).
Point mutations of the xcex2APP gene are linked to a relatively small number of FAD pedigrees such as xcex2APP-London, xcex2APP-Flemish, and xcex2APP-Swedish (Goate, A. M., et al., 1991 Nature 349:704-706; Chartier-Harlin, M. -C., et al., 1991 Nature 353:844-846; Murrell, J., et al., 1991 Science 254:97-99; Karlinsky, H., et al., 1992 Neurology 42:1445-1453; Mullan, M., et al., 1992 Nature Genetics 1:345-347). Point mutations of the PS2 gene are also linked to a minority of FAD cases (Levy-Lahad, E., et al., 1995 Science 269:973-977; Rogaev, E. I., et al., 1995 Nature 376:775-778). The majority of FAD cases are caused by point mutations of the PS1 gene (Sherrington, R., et al., 1995 Nature 375:754-760), which results in a selective increase of the Axcex2-42 peptide (Scheuner, D., et al., 1996 supra).
PS1 and PS2 are integral-membrane proteins, having 6 or 8 transmembrane domains (Doan, A., et al., 1996 Neuron 17:1023-1030; De Stooper, B., et al., 1997 supra), and are located in the endoplasmic reticulum, early Golgi, and possibly at the cell surface (Xia, W., et al., 1998 Biochem. 37:16465-16471; Kovacs, D. M., et al., 1996 Nature. Med. 2:224-229; Ray, et al., 1999 J. Biol. Chem. 274:36801-36807). These presenilin proteins share 63% sequence identity.
It has been postulated that PS1 may be the elusive gamma-secretase. Evidence to support this postulate includes the observation that cells from PS1-deficient mouse embryos generate significantly reduced levels of the Axcex2 peptide, demonstrating that PS1 appears to play a role in facilitating gamma-secretase activity (De Stooper, B., et al., 1997 supra). In particular, it is postulated that PS1 is an autoactivated aspartyl protease having gamma-secretase activity (Wolfe, M. S., et al., 1999 Nature 398:513-517). This hypothesis is based on the discovery that two aspartate residues, which reside within the transmembrane domain of PS1, are required for endo-proteolytic processing of PS1 and gamma-secretase activity (Wolfe, M. S., et al., 1999 supra). Point mutations of residues aspartic acid-257 to alanine or aspartic acid-385 to alanine inhibited endo-proteolysis of PS1, and caused an accumulation of the C100 and C83 APP fragments, suggesting that these aspartate residues are required specifically for gamma-secretase activity. Similar results have been reported for mutant PS2 proteins which contain point mutations of the corresponding aspartyl residues (Kimberley, W. T., et al., 2000 J. Biol. Chem. 275:3173-3178). Yet there is no evidence that PS1 or PS2 directly catalyzes cleavage of a xcex2APP substrate. Furthermore, PS1 and PS2 lack sequences and structural similarity with known proteases and aspartyl proteases.
An alternative hypothesis suggests that PS1 functions as a regulatory cofactor of the xcex2APP cleavage pathway (De Stooper, B., et al., 1997 supra; Wolfe, M. S., et al., 1999 Nature 398:513-517). Support for this hypothesis comes from the observation that PS1 shares structural similarity with SREBP cleavage-activating protein (SCAP) which is also an integral-membrane protein having 6 to 8 transmembrane domains and plays a role in regulating cleavage of SREBP (Hua, X., et al., 1996 Cell 87:415-426; Brown, M. S. and Goldstein, J. L. 1997 Cell 89:331-340; Sakai, J., et al., 1997 J. Biol. Chem. 272:20213 20221).
The hypothesized roles of the presenilins and gamma-secretase are further complicated by the fact that C-terminal cleavage products of a xcex2APP-like protein, the APLP1 protein (Wasco, W., et al., 1992 Proc. Natl. Acad. Sci. USA 89:10758-10762), accumulate in primary neurons that lack PS1 (Naruse, S., et al., 1998 Neuron 21:1213-1221). One hypothesis that explains this result is that PS1 modulates trafficking of the C-terminal fragments that result from cleavage of the xcex2APP and APLP1 proteins (Naruse, supra).
The possible role of presenilins and gamma-secretase also extends to proteolytic processing of proteins other than xcex2APP and xcex2APP-like proteins. For example, it has been previously determined that the presence of PS1 is required for proteolytic cleavage of the Notch protein, which is a single transmembrane domain cell surface receptor that mediates many cell fate decisions in vertebrates and invertebrates (Artavanis-Tsakonas, S., et al., 1996 Science 268:225-232; Kopan, R. and Turner, D. 1996 Curr. Opin. Neurobiol. 6:594-601; Weinmaster, G. 1997 Mol. Cell. Neurosci. 9:91-102). Mutations of the two transmembrane aspartate residues within PS1 inhibits cleavage of Notch proteins (Ray, W. J., et al., 1999 J. Biol. Chem. 274:36801-36807). The postulated gamma-secretase cleavage sequence within an S2-cleaved Notch-1 protein (Schroeter, E. H., et al, 1998 Nature 393:382-386) has no similarity with commonly accepted protease cleavage site motifs.
The role of the presenilins and gamma-secretase can be settled by isolating a protein or a protein complex having the functional-activity of gamma-secretase. In general, it is difficult to isolate functionally-active integral-membrane proteins and protein complexes, as they tend to lose their functional activity during the isolation procedure. This difficulty has been overcome by the development of various methods that are described herein.
In addition, the present invention provides an isolated protein complex having gamma-secretase activity. The isolated gamma-secretase protein complex of the invention catalyzes cleavage of polypeptide substrates having gamma-secretase cleavage sequences. It is postulated that the gamma-secretase protein complex of the present invention is the putative gamma-secretase which is responsible for the processing pathway that generates the Axcex2-42 peptide.
As a preliminary matter, the detection of gamma-secretase activity requires assays capable of reliable, accurate and expedient detection of the presence or absence of gamma-secretase cleavage products. Moreover, where inhibitors of gamma-secretase activity are desired, it would be particularly helpful to accurately screen a large volume of test compounds without undue processing.
The present invention therefore provides homogenous methods for detecting gamma-secretase activity and inhibitors thereof. The discovery and application of homogenous assay methods for gamma-secretase activity allows for detection of activity without necessitating the steps of isolating and retrieving gamma-secretase cleavage products. The elimination of these steps, for isolating and retrieving cleavage products, provides obvious benefits in terms of speed and accuracy. In addition, the present invention provides homogenous methods for detecting specific products of gamma-secretase activity, including the detection of Axcex2 or the 6 kDa fragment.
The present invention provides the discovery that gamma-secretase is an integral membrane protein that is found in the endoplasmic reticulum, Golgi apparatus, and plasma membrane of various mammalian cell types.
The present invention provides an isolated protein that catalyzes the proteolytic cleavage of a substrate, such as a xcex2APP polypeptide; the functionally-active protein complex is described herein as a gamma-secretase, e.g., a gamma-secretase complex. The present invention provides an isolated cell-free membrane fraction which includes functionally active gamma-secretase. The present invention also provides a gamma-secretase protein complex that is isolated in a solubilized form.
The present invention provides methods for isolating the gamma-secretase protein by co-isolating it with PS1. Additionally, the present invention provides methods for isolating solubilized integral-membrane proteins or protein complexes, such as the gamma-secretase complex.
In addition, the present invention provides a composition, comprising N-3[(dimethylamino) propyl]3,7, 12-trihydroxy (3a,5b,7a,12a) cholan-2-amide] and CHAPSO(trademark); the novel composition is useful for isolating the gamma-secretase protein complex, reconstitution methods, isolating a substrate, and identifying reagents that inhibit gamma-secretase activity.
The present invention also provides methods for detecting gamma-secretase activity and for detecting the production of gamma-secretase products, particularly, Axcex2. In addition, the present invention provides methods for identifying reagents that inhibit gamma-secretase activity.
To identify gamma-secretase inhibitors, a test compound is introduced to a sample containing uncleaved xcex2APP, xcex2APP fragments, and gamma-secretase. The gamma-secretase is activated and the effect of the test compound on the amount of gamma-cleaved xcex2APP fragment produced is monitored. Where xcex2-secretase has cleaved fragments or is also present, the amount of Axcex2 can be monitored.
In particular, the present invention provides an efficient system for detecting the cleavage of xcex2APP substrates by gamma-secretase in fluid samples, namely by measuring the production of gamma-cleaved xcex2APP fragments. The detection system utilizes a pair of fluorescent adducts which are capable of transferring fluorescent energy from one to the other. By using the pair as labels for the substrates and products of gamma-secretase, the activity of gamma-secretase can be monitored.
The binding assay operates by binding each of the fluorescent adducts as labels to different portions of the same gamma-cleaved xcex2APP fragment. In a preferred embodiment of the invention, the first of the fluorescent adducts binds specifically to the carboxy terminal end of a gamma-cleaved xcex2APP fragment, at the site of normal gamma-secretase cleavage, i.e., at amino acid residue 711 (corresponding to Axcex2 amino acid residue 40), while the second fluorescent adduct binds to a portion of the same gamma-cleaved xcex2APP fragment in the amino terminal region, in amino acids 1 through 702. Most preferably, particularly where Axcex2 detection is also an objective, the second fluorescent adduct binds within an amino acid sequence corresponding to amino acid sequence 1-31 of Axcex2. Optionally, it can be conceived that the first fluorescent adduct may instead specifically bind to the carboxy terminal end of a gamma-cleaved xcex2APP fragment at amino acid 713 (Axcex2 amino acid residue 42), the cleavage site most commonly associated with mutations in xcex2APP, PS1 or PS2. Preferably, the fluorescent adducts do not bind to overlapping sites of the gamma-cleaved xcex2APP fragment, and the first fluorescent adduct, which is specific to the gamma-cleaved xcex2APP at its carboxy terminal end, has substantially no cross-reactivity to either uncleaved xcex2APP or to other types of gamma-cleaved xcex2APP fragments. Gamma-secretase cleavage is detected when excitation of one of the bound fluorescent adducts provides a detectable transfer of energy to the other fluorescent adduct.
In an alternative embodiment for the detection of gamma-secretase cleavage, the adducts bind to separate cleavage products. Each of the fluorescent adducts would bind to separate amino acid sequences corresponding to opposite sides of the gamma-secretase cleavage site on an uncleaved xcex2APP. Preferably in this alternative embodiment, at least one of the fluorescent adducts binds to its amino acid sequence with substantially no cross-reactivity to other portions of uncleaved xcex2APP. Where gamma-secretase cleavage has occurred, the fluorescent adducts would each be bound to their separate gamma-cleaved xcex2APP fragments, thus resulting in a substantially decreased transfer of energy upon excitation.